Cell-free sensor systems
AssigneeenEvolv, Inc.
InventorsJames E. SPOONAMORE, Matthew R. DUNN, Noah D. Taylor, Kristin J. ADOLFSEN, IIan N. WAPINSKI, Jay H. KONIECZKA
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Abstract
The present described inventions relate, inter alia, to methods and compositions that provide for improved detection of target molecules in, for example, bioengineering.
Claims55
A method of making an allosteric DNA-binding protein sensor and/or switch which binds to a target molecule, comprising:
- (a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a DNA-binding protein sensor and/or switch for an ability to bind a target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield a candidate allosteric DNA-binding protein sensor and/or switch having an ability to bind a target molecule;
- (b) providing a host cell with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and a nucleic acid encoding a reporter gene system and selecting for a cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system;
- (c) isolating nucleic acids from the cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture, the IVT or IVTT mixture comprising a target molecule and a detection reagent;
- and
- (d) interrogating the IVT or IVTT mixture for reporter response, the reporter response being indicative of target molecule binding to the candidate allosteric DNA-binding protein sensor and/or switch.
The method of any one of the above claims, wherein the target molecule is an antibiotic.
The method of any one of the above claims, wherein step (a) comprises mutating an allosteric protein.
The method of any one of the above claims, wherein the nucleic acid is provided to the host cell by one or more of electroporation, chemical transformation, ballistic transformation, pressure induced transformation, electrospray injection, mechanical shear forces induced, for example, in microfluids, and carbon nanotubes, nanotube puncture, induced natural competence mechanisms of an organism, merging of protoplasts, and conjugation with Agrobacterium.
The method of any one of the above claims, wherein the host cell is selected from a eukaryotic or prokaryotic cell, selected from a bacterial, yeast, algal, plant, insect, mammalian cells, and immortalized cell.
The method of any one of the above claims, wherein the reporter gene system comprises a protein having a unique spectral signature and/or assayable enzymatic activity.
The method of any one of the above claims, wherein the IVT or IVTT mixture comprises a coupled or linked system.
The method of any one of the above claims, wherein the reporterresponse is a direct amplification of the genotype of the allosteric protein.
The method of any one of the above claims, wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the nucleic acid encoding the reporter gene system comprises a single nucleic acid vector.
The method of any one of the above claims, wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the nucleic acid encoding the reporter gene system comprises two nucleic acid vectors.
The method of any one of the above claims, further comprising:
- (e) isolating the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch.
A method of making an allosteric DNA-binding protein sensor and/or switch which binds to a target molecule, comprising:
- (a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a DNA-binding protein sensor and/or switch for an ability to bind a target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield a DNA-binding protein sensor and/or switch which has an ability to bind a target molecule;
- (b) providing a host cell with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and a nucleic acid encoding a reporter gene system and selecting for a cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system;
- (c) isolating nucleic acids from the cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture, the IVT or IVTT mixture comprising a target molecule and a detection reagent;
- and
- (d) interrogating the IVT or IVTT mixture by nucleic acid sequencing before and after selection to determine those molecules that have become functionally enriched.
A method of making an allosteric DNA-binding protein sensor and/or switch which binds to a target molecule, comprising:
- (a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a DNA-binding protein sensor and/or switch for an ability to bind a target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield the candidate allosteric DNA-binding protein sensor and/or switch having an ability to bind a target molecule;
- (b) contacting a solid support with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and selecting for a solid support comprising the candidate allosteric DNA-binding protein sensor and/or switch;
- (c) isolating nucleic acids from the solid support comprising the candidate allosteric DNA-binding protein sensor and/or switch and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture;
- (d) introducing a reporter gene system, detection reagent, and target molecule, and interrogating the mixture for a reporter response, the reporter response being indicative of the target molecule binding to the candidate allosteric DNA-binding protein sensor and/or switch.
A method for making a target molecule in a biological cell, comprising:
- (a) engineering the biological cell to produce the target molecule;
- (b) introducing an allosteric DNA-binding protein sensor and/or switch which binds to the target molecule in the biological cell;
- and
- (c) screening for target molecule production.
Dependencies are inferred from the claim text.